Interconnected Porous Calcium Hydroxyapatite Research Articles

The Effect of Simulated Microgravity by Three-Dimensional Clinostat on Bone Tissue Engineering

Evidence suggests that mechanical stress, including gravity, is associated with osteoblast differentiation and function. To examine effects of microgravity on bone tissue engineering, we used a three-dimensional (3D) clinostat manufactured by Mitsubishi Heavy Industries (Kobe, Japan). A 3D clinostat is a device that generates multidirectional G force. By controlled rotation on two axes, it cancels the cumulative gravity vector at the center of the device. We cultured rat marrow mesenchymal cells (MMCs) in the pores of interconnected porous calcium hydroxyapatite (IP-CHA) for 2 weeks in the presence of dexamethasone using the 3D clinostat (clinostat group). MMCs cultured using the 3D clinostat exhibited a 40% decrease in alkaline phosphatase activity (a marker of osteoblastic differentiation), compared with control static cultures (control group). SEM analysis revealed that although there was no difference between the two groups in number or distribution of cells in the pores, the clinostat group exhibited less extensive extracellular matrix formation than the control group. Cultured IP-CHA/MMC composites were then implanted into subcutaneous sites of syngeneic rats and harvested 8 weeks after implantation. All implants showed bone formation inside the pores, as indicated by decalcified histological sections and microfocus computed tomography. However, the volume of newly formed bone was significantly lower for the clinostat group than for the control group, especially in the superficial pores close to the implant surface. These results indicate that new bone formation in culture was inhibited by use of the 3D clinostat, and that this inhibition was mainly due to suppression of osteoblastic differentiation of MMCs.

Bone tissue engineering using novel interconnected porous hydroxyapatite ceramics combined with marrow mesenchymal cells: quantitative and three-dimensional image analysis.

We developed fully opened interconnected porous calcium hydroxyapatite ceramics having two different pore sizes. One has pores with an average size of 150 microm in diameter, an average 40-microm interconnecting pore diameter, and 75% porosity (HA150). The other has pores with an average size of 300 microm in diameter, an average 60-100-microm interconnecting pore diameter, and 75% porosity (HA300). Because of its smaller pore diameter, HA150 has greater mechanical strength than that of HA300. These ceramics were combined with rat marrow mesenchymal cells and cultured for 2 weeks in the presence of dexamethasone. The cultured ceramics were then implanted into subcutaneous sites in syngeneic rats and harvested 2-8 weeks after implantation. All the implants showed bone formation inside the pore areas as evidenced by decalcified histological sections and microcomputed tomography images, which enabled three-dimensional analysis of the newly formed bone and calculation of the bone volume in the implants. The bone volume increased over time. At 8 weeks after implantation, extensive bone volume was detected not only in the surface pore areas but also in the center pore areas of the implants. A high degree of alkaline phosphatase activity with a peak at 2 weeks and a high level of osteocalcin with a gradual increase over time were detected in the implants. The levels of these biochemical parameters were higher in HA150 than in HA300. The results indicate that a combination of HA150 and mesenchymal cells could be used as an excellent bone graft substitute because of its mechanical properties and capability of inducing bone formation.